Liquid chromatography mass spectrometry (“LCMS”) is a powerful analyte detection and measurement technique that has become the preferred method of detecting small molecule, amino acid, protein, peptide, nucleic acid, lipid, and carbohydrate analytes to a high accuracy for diagnostic purposes. The chromatographic separation process relies on the fact that a number of component solute molecules in a flowing stream of a fluid percolated through a packed bed of particles, known as the stationary phase, can be efficiently separated from one another. Generally, separation in liquid chromatography is achieved in a column by selective distribution of the sample molecules between a stationary phase and a mobile phase. The individual sample components are separated because each component has a different affinity for the stationary phase, leading to a different rate of migration for each component and a different exit time for each component emerging from the column. The separation efficiency is determined by the amount of spreading of the solute band as it traverses the bed or column.
Reversed-phase liquid chromatography (RPLC) is widely used as a mode of separation in chromatographic systems. In the RPLC technique, the solvent(s) employed in the mobile phase is/are more polar than the stationary phase, whereas the reverse situation is true in conventional (normal phase) chromatography. The mobile phase solvents typically employed in reversed phase liquid chromatography systems comprise water and one or more water-miscible organic modifiers, for example, acetonitrile or methanol. Analyte species of-interest typically form a solution with the mobile phase. The RP-HPLC stationary phase is usually highly hydrophobic or non-polar. The affinity of a chemical species for a stationary phase, which affects the rate at which the particular species in a flowing mobile phase passes through the stationary phase, results primarily from interaction of the species with chemical groups present on the stationary phase. These chemical groups may be provided on the stationary phase by reacting a surface-modifying reagent with a substrate, such as a silica substrate. Surface-modifying agents may thus be employed to adsorb specific chemical groups onto the stationary phase. Conventional reversed-phase liquid chromatography uses 1.5-10 μm spherical silica beads that have been modified by covalent attachment of hydrocarbon chains including 4, 8, or 18 carbon atoms to provide a non-polar surface.
Clinical laboratories routinely measure the concentrations of drug compounds (including pharmaceutical compounds as well as other natural and synthetic drugs of abuse) in human-provided body fluids. These compounds generally comprise low molecular weight molecules that are very hydrophilic and difficult to retain on most reversed phase LC systems. The metabolites are even more hydrophilic and are even harder to retain on reverse phase LC systems. Alternative HPLC systems, such as those employing HILIC columns, retain the metabolites but not all of the parent compounds. Thus, quantification of drug compounds using chromatographic separation techniques is quite challenging. For this reason, current clinical quantification of drugs of abuse is carried out by hydrolysis of the phase two metabolites and some phase one metabolites (either by acid or enzymatic hydrolysis) in order to convert the metabolite back to the parent compound and subsequently, LCMS measurement of only the parent compound is necessary. The total concentration of the metabolites and parent drug are reported together as a single value because the metabolites have been converted back to the parent during hydrolysis. Either acid hydrolysis or enzymatic hydrolysis requires a treatment step followed by up to 2 hours of incubation at an elevated temperature. Thus, the hydrolysis procedure adds extra time and cost to each analysis.
In accordance with the above discussion, there is a need in the art for a quick and reliable chromatographic separation and analysis methods—such as an LCMS method that does not require an additional hydrolysis step—for routine clinical measurements of drug compounds. There is also a need for new chromatographic designs that are able to implement the new methods. Unfortunately, the conventional instrumentation required for LCMS is technically complex and not well suited to the typical hospital clinical lab or medical lab technician. These clinical labs have not adopted LCMS diagnostics and, instead, generally use alternative diagnostic techniques, including automated immunoassay. Alternatively, the clinical labs may send the samples out to a central reference laboratory for analysis.
Recently, however, an automated analyzer that is suitable for routine clinical and hospital use has recently been described in international patent application (PCT) publication WO 2012/058632 A1 titled “Automated System for Sample Preparation and Analysis” which is hereby incorporated by reference herein in its entirety. Adaptation of such an automated analyzer so as to further include routine analyses for drugs of abuse using one of the conventional hydrolysis methods would require either off-line hydrolysis followed by transfer of the treated samples to the automated analyzer or else on-board automated robotic hydrolysis. Implementation of either of these modifications would require additional hardware and increased overall system complexity. Further, a recent study (Wang et al., “Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites”, Journal of Analytical Toxicology, Vol. 30, October 2006, pp. 570-575) has concluded that, with regard to at least opioids in urine samples, acid hydrolysis liberates a greater proportion of the parent drug compounds and introduces less variability than enzymatic hydrolysis. To implement the evidently preferred method of acid hydrolysis for such samples would require additional expensive safeguards to prevent lab personnel from being exposed to hazardous reagents. Therefore, there is a special need in the art for a quick and reliable chromatographic separation and analysis method—such as an LCMS method that does not require an additional hydrolysis step—that may be employed using an automated clinical analyzer to make routine clinical measurements of drug compounds.